Isolation of vasoactive intestinal peptide

ABSTRACT

A polypeptide having biological action including systemic vasodilation, hypotension, increased cardiac output, respiratory stimulation, and hyperglycemia isolated from intestines of animals such as a hog. The isolation procedure for purification includes two steps of ion-exchange chromatography, gel chromatography and countercurrent distribution separation. The polypeptide, VIP, has the amino acid structure: His (Ala, Asp, Ser, Val). Phe, Thr. (Asx2). Tyr. Thr. Arg. Leu. Arg. (Lys, Glx). Met. Ala (Val, Lys, Lys). Tyr. Leu. Asn. Ser. Ile. Leu. Asn. NH2. Therapeutic biological actions may be produced in animals and humans by intravenous injection by .02-10 Mu g VIP per kg of body weight.

United States Patent 1191 Said et al.

Sept. 17, 1971 (Under Rule 47) 1 App]. No.: 181,444

[22] Filed:

[52] US. Cl 260/1125; 424/177 [51] Int. Cl.. C07c 103/52; C07g 7/00;Aolk 27/00 [58] Field of Search 260/1 12.5

[56] References Cited OTHER PUBLICATIONS Said et al., Nature, 225, 863(Feb. 28, 1970). .lorpes et al., Acta Chem. Scand., 18, 2408 (I964).Said et al.. Nature. 224, 699 (1969). Mutt et al.. Rec. Prog. HormoneRes, 23, 483 (1967).

[ Apr. 22, 1975 Haefely et al.. Experientia. 18, 297 (1962).

Prirmzry E.\'uminet-Lewis Gotts Assistant E.\'aminerReginald J. SuyatAttorney. Agent. or Firm-Lowe, King & Price [57] ABSTRACT A polypeptidehaving biological action including systemic vasodilation. hypotension.increased cardiac output. respiratory stimulation. and hyperglycemiaisolated from intestines of animals such as a hog. The isolationprocedure for purification includes two steps of ion-exchangechromatography, gel chromatography and countercurrent distributionseparation.

The polypeptide. VIP, has the amino acid structure: His (Ala, Asp. Ser.Val). Phe, Thr. (Asx. Tyr. Thr. Arg. Leu. Arg. (Lys, Glx). Met. Ala(Val. Lys, Lys). Tyr. Leu. Asn. Ser. Ile. Leu. Asn. NH.

Therapeutic biological actions may be produced in animals and humansintravenous injection by .02-10 [.Lg VIP per kg of body weight.

6 Claims, 4 Drawing Figures Pmimznwazzma 3,879,371

SEEKET 1 BF 2 FIRST CHROMATOGRAPHY ON CARBOXYMETHYL CELLULOSE (Buffers:O .O I25MSodium orthophosphure, OZMHCL) OPTICAL DENSITY 280 m 1 |.O-SECRETIN --g i i l O 3 'e 1 3 T8 FRACTION NUMBER CHROMATOGRAPHY ONSEPHADEX (-25 (Buffer,0.2MAceflc acid) OPTICAL DENSITY 280 mp FRACTIONNUMBER FIGZ so -"4s 3o FEHEL-mazzmra 79,371

sum 2 or g COUNTER -CURRENT DISTRIBUTION loo (l-Butanol/OIMAmmoniumbicarbonate) OPTICAL 0.6- DENSITY 2nd. CHROMATOGRAPHY .ON. CARBOXYMETHYLCELLULOSE (Buffer, 0.1MAmmonium bicarbonate) OPTICAL DENSITY 280 mp I9 24 2'9 3'4 3'9 4'4 4'9 5'4 5'9 64 69 74 7584 as 94 FRACTION NUMBER FIG. 3

ISOLATION OF VASOACTIVE INTESTINAL PEPTIDE This invention relatesgenerally to a new polypeptide, its method of isolation, biologicalactions, and therapeutic usefulness. More particularly, the presentinven tion relates to new polypeptide isolated from the intestines ofmammals by means of new purification procedures and the wide-range ofactivity of the polypeptide affecting cardiovascular, respiratory andmetabolic functions.

It is well-known that polypeptide hormones regulate many physiologicfunctions and mediate certain pathological responses. While numerouscompounds have been known to possess a vasodilator effect, many possessthis effect only to a relatively slight degree or for a very short time.Medical science has, therefore, sought materials exhibiting a morepotent or sustained vasodilator effect and which would be more usefultherapeutic agents.

It is the principal object of the present invention to provide a newpolypeptide exhibiting such biological actions.

It is also an object of the present invention to provide a newpolypeptide exhibiting activity as a systemic vasodilator, hypotensive,cardiac output increaser and respiratory stimulator as well as aproducer of hyperglycemia.

This invention also has as an object the provision of separatingtechniques for isolating the polypeptide from intestines of mammalsthrough the use of chromatography and other separation procedures.

This invention also has as an object the provision of new polypeptidehaving a known amino acid structure which exhibits unexpected biologicalactivity.

A further object of the present invention is a provision of a method oftreating humans and animals by intravenous injection of the newpolypeptide.

These and other objects of the present invention will become apparentfrom careful consideration of the following specification and claimsincluding the drawings wherein:

FIGS. 1 through 4 illustrate the plottings of optical density versusfraction number as obtained in accordance with the variouschromatography and countercurrent distribution procedures.

Secretin has been obtained from hog intestines in accordance with amethod discovered by one of the coinventors of this invention,Preparation of Highly Purified Secretin, Arkiv f. Kemi l5, 8 (I959). Thesame procedure provides the starting material for this invention.

It should be understood that the starting material for the presentinvention contains secretin and VIP (vasoactive intestinal polypeptide)in accordance with the Mutt procedure outlined in the above-citedpublication is only typical of the material that may be used as thestarting material. Any procedure that extracts secretin and VIP havinghigh vaso activity would be suitable as starting material. The startingmaterial, though not the only one acceptable, would be that as outlinedin the above-mentioned article through page 72, line ll.

In the article by the present inventors in Nature 225,5235, Pg. 863-864,Feb. 24, 1970, the starting material obtained from the Mutt procedurewas chromatographed on carboxymethyl cellulose (CMC). Furtherpurification was obtained through gel chromatography and the column ofSephadex. It was presumed that the product from these two chromatographyseparations would be sufficient to obtain a purified product. However,further experimentation led to the discovery that special purificationprocedures were necessary since the product disclosed in the Naturearticle was found not to be pure.

It was discovered that the crude VIP and secretin must first besubjected to a first ion-exchange chromatography followed by separationby gel chromatography, thereafter followed by a second ion exchangechromatography separation and finally a countercurrent distributionseparation.

The first ion-exchange chromatography separation as well as the secondion-exchange chromatography separation utilize CMC, though otherion-exchange media may be utilized. The buffer may be the sodium orammonium (carbonate), bicarbonate. phosphate or acid phosphate. Theconcentration of the buffer is not critical but preferably may varybetween approximately .01 and .0l5M. The pH of the buffer solutionshould be between 7 and 9, preferably between 7.8 and 8.2. The crudepolypeptide containing secretin and VIP is dissolved in the buffer andpassed onto the column of CMC. FIG. 1 illustrates the optical density at280 pg of the various fractions collected. Fractions 9 through 18contain the bulk of the secretin activity. All of the vaso activity ofthe various fractions throughout this invention were determined byinfusing various fractions into the femoral artery of the dogs leg, andmeasuring the blood flow in that artery.

The fractions containing crude VIP include those fractions beginningapproximately after the last of the secretin has been removed, and inany event those fractions having an optical density within the firstpeak after the passage of the secretin. In the example, fractions 20through 25 are collected to obtain all of the VIP when eluted with anacid, particularly hydrochloric acid. The obtained VIP is then convertedto the acetate and freeze-dried.

After the first ion-exchange separation made on the basis of the acidityor basicity of the polypeptides, a gel chromotography separation isutilized to separate the various polypeptide molecules in accordancewith their size. The column may be Sephedex, which is a wellknowncross-linked dextran frequently used for this purpose, or cellulose or alike gel filtration medium. The crude VIP from the previous and firstionexchange chromatography is dissolved in an organic acid such asacetic acid. The concentration of the acid is not critical but may varyfrom approximately 0.1M to 0.3M. The preferred organic acid is aceticacid. FIG. 2' illustrates the optical density of the fractions at 280 a.The fractions should be collected at approximately the highest portionsof the peak of the optical density curve. In the example, fractions 43through 48 were collected and found to contain the crude VIP.. Thesefractions were combined and freeze-dried.

The crude VIP contained in the freeze-dried product was now ready forthe second ion-exchange chromatography on CIMC. The crude VIP wasdissolved in am monium bicarbonate and the pH adjusted between pH 7-9,preferably pH 7.8-8.2.

The concentration of the buffer may be 0.05M to 0.15M approxmately; aspreviously stated, the concentrations are not critical.

The fractions that were obtained exhibited an optical density as shownin FIG. 3. By testing the vaso activity, there was discovered that thefractions should be collected beginning at about 1/4 to H5 of the peakvalue of the optical density down to approximately l/ of the peak value.The crude VIP so obtained from the fractions are shown to be fractions61 through 83 and were combined and freeze-dried.

The final essential purification was discovered to be a countercurrentdistribution separation. The systems preferred included an organicsolvent, for example: alcohols such as methanol and l-butanol and anaqueous solvent, preferably alkali metal, salts of chlorides,bicarbonates, phosphates, etc; for example, sodium chloride, potassiumphosphates and ammonium bicarbonate. At least a 20 tube separationsystem should be used, but also a several hundred tube system may beused, or even greater.

Concentration of the ammonium bicarbonate may vary from 0.05M to 0.15M.Again, the concentrations are not critical.

The collections were made on the basis of the optical densities of thehomogenous tube contents as deter- ,mined at 215p" Collections were madeof those frac- EXAMPLE PRELIMINARY SEPARATION The uppermost metre of hogintestine was removed from the animals as soon as possible, cleansed ofthe bulk of adhering fatty tissue, emptied of its'contents and flushedwith cold water. ltwas then immersed, without being everted, for 5-10minutes in vigorously boiling water. The boiled intestines were storedat about -l-5C for not longer than one month. They were worked 'up inbatches of 1000 intestines each. The intestines in such a batch weightedabout 70 kg.They were minced in the frozen state and extracted withconstant stirring overnight at room temperature with 200 litres of 0.5 Nacetic acid. Tap water was used for diluting the glacial acetic acid.Two kg of Hyflo SuperCel was then added to the extraction mixture, andthe resulting suspension filtered through bags of linen cloth. It wasstirred with 2 kg of alginic acid, which had previously been washed withwater, 0.2 N HCl, and water again. The alginic acid containing theadsorbed VIP was allowed to sediment for a few hours. The-supernatantwas discarded, and the sediment transferred to suction filters, on whichit was washed with 0.005 N HCl and then with 95 percent ethanol toremove the fats. The ethanol was either allowed ito evaporate, or washedoff with 0.005 N HCl. TheVlP was eluted from the alignic acid with 20litres of 0.2 N HCl under stirring for one hour. After filtering, theactive material was precipitated from the eluate with sodium chloride atsaturation. The precipitate was collected on a suction filter and suckedas dry as possible.

The precipitate was dissolved in water at room temperature to aconcentration of 5 g per 100 ml solution. Two volumes of percent ethanolwere added and the pH of the solution, determined electrometrically,brought to 7.2 with a mixture of one part of 1 N NaOH and two parts of95 percent ethanol. The precipitate that formed was filtered off onfluted paper (Whatman 3MM) and discarded. The clear filtrate was dilutedwith an equal volume of 0.15 percent acetic acid, g of alginic acid,which had been prewashed with 0.2N HCl and then with water, wassuspended in the diluted filtrate. After stirring for one hour at roomtemperature, the alginic acid containing the adsorbed VIP was collectedon a suction filter and washed on the filter with 0.005N HCl. Thefiltrate and washings were discarded. Elution of the VIP from thealiginic acid was carried out under stirring for 10 minutes with 1.5litres of 0.2N HCl. The alginic acid was filtered off and washed withwater. The eluate was saturated with sodium chloride. The precipitatethat formed was collected on a suction filter. lt weighed 20.5 g andcontained 9.0 percent N. The precipitate was dissolved in water to aconcentration of 5 'g per 100 ml. and the pH of the solution brought to7.2 with 0.1N NaOH. A precipitate formed 2 g of Hyflo Super-Cel,prewashed with 2N HCl and water and dried at C, were added to every 100ml of the solution, and the mixture filtered with suction. The filtercake was discarded, the clear filtrate brought to pH 4.5 with. N HCl,and saturated with sodium chloride. The precipitate that formed wascollected on a suction filter. It was covered on the filter with a layerof soft plastic and sucked as dry as possible.

This precipitate was triturated for 15 minutes at +4C with 50 ml ofmethanol per g precipitate. The suspension was filtered on a suctionfilter. The undissolved material, which contained cholecystokinin andpancreozymin, was washed on the filter with methanol and ether. Theair-dry material weighed 5g. It was stored for later use. The filtratewas brought to about pH 7.5 (glass electrode) with 0.1 M NaOH inmethanol. The precipitate that formed was filtered off on fluted paperand discarded. The filtrate was brought to pH 6.0 with 0.1 M HCl inmethanol, and precipitated with 2 volumes of ether precooled to 15C. Theprecipitate that formed was collected at l5C on a suction filter, and

'washed on the filter with ether. It was then dissolved to a 2 percentsolution in water, and the solution saturated with sodium chloride. Theprecipitate was collected on a suction filter on hardened paper, whichhad been moistened with a saturated solution of sodium chloride inwater.

This precipitate constitutes the starting material for the subsequentseparation techniques. As outlined above, the method follows preciselythat disclosed by Mutt for the purification of secretin.

CHROMATOGRAPHIC SEPARATION The first CMC column.

450 g of CMC was washed sequentially with ethanol, water, 0.2 M HCl and0.1 M NaOH. It was then suspended in 10 liters of the latter solutionand allowed to sediment for one hour. The supernatant, containing fineswas poured off and the procedure of suspending and decanting repeatedonce. The CMC was washed again with 0.2 M HCl and, suspended in this,was poured into a chromatographic tube, 14x40 cm. The glass tube, madeby Messrs. Werner-Glas, Stockholm, had a flat, perforated bottom whichwas covered by a layer of nylon netting and then cloth. The netting andcloth were held in position by a disc of perforated tefion, fittingfirmly into the tube. After setting, the top of the column was coveredwith a layer of nylon cloth, held in position by a perforated porcelaindisc. To prevent toppling, a glass rod, directed vertically upwards, wasattached to the middle of the disc, and to this rod two other rods, eachslightly shorter than the diameter of the tube, were attached crosswise,about ten cms above the plane of the disc. The column was washed with0.1 M NaOH and kept under this solution until required forchromatography.

First Chromatography on CMC The column was washed with a 0.0125 M sodiumorthophosphate buffer of pH 8- -0.l; containing EDTA and tricresol. Thecomposition of the buffer was: 44,77 g Na HPO .l2H O, 3.70 g disodiumEDTA and 20 ml tricresol (distilled, colorless), in water to a volume often liters. Before use, the buffer was clarified by filtration throughMillipore filters of 0.45U porosity. The column was usually kept underthis buffer overnight. It was then equilibrated with a buffer containingthe same amount of disodium orthosphosphate in ten liters but with theEDTA and tricresol replaced by 500 ml of dimethyl formamide. The pH wasadjusted to 8:t0.l by the addition of about 7 ml of l M H PO Thisbuffer, too, was filtered immediately before use. After equilibration,the column had the dimensions 14X14 cm. 12 g of the starting materialwas dissolved in 480 ml of buffer and the pH of the solution was broughtto 810.1 by

the addition of about 590 ml of 0.02 M NaOH which contained 50 ml/literof dimethyl formamide. A slight precipitate formed. It was filtered offon fluted Munktell 00 paper with the aidof 20 g of Hyflo Super Cel anddiscarded. The clear solution was allowed to sink into the column andthe chromatogram was developed with the buffer at a flow rate of 100ml/min. Fractions of 500 ml each were collected. After the 18 thfraction, starting with the introduction of the polypeptide solutioninto the column, the eluant was changed to 0.2 M HC1 and more fractionswere collected. FIG. 1 shows the optical density at 280 p. of thefractions. Fractions 9-18 contained all of the secretinactivity.Fractions 22-25 were obtained after all fractions containing secretinwere first obtained as illustrated in FIG. I. These fractions 22-25 werecombined and diluted to 20 liters with water. 120 g (wet weight) ofalginic acid was suspended in the solution and the mixture stirred forminutes. The alginic acid, carrying the'absorbed polypeptide material,was allowed to settle and was then collected on a suction filter andwashed thoroughly with ice-cold 0.005 M HCl. The polypeptides wereeluted with 900 ml of ice-cold 0.2 M HCl, added in portions of 100 mleach. The eluate, followed by 0.2 M acetic acid, was passed through a6X17 cm column of DEAE-Sephadex in acetate form and thepolypeptidecontaining, choloride-free effluent was lyophilized. Thelyophilized material weight 1.9 g.

The SephadexG-ZS column.

Sephadex G-25 fine, was washed sequentially with ethanol, 0.1 M NaOH,0.2 M HCl, and 0.2 M acetic acid containing 3 ml/liter of tricresol. Aquantity of sephadex, suspended in the latter solution, and sufficientto give an 80 cm column after settling, was poured into a 10X 100 cmglass tube fitted with a packing reservoir (Pharmacia K 10/100 and R100). The column was stored under the tricresol-containing acetic aciduntil required for chromatography.

Chromatography on Sephadex G-25.

The column was washed with 0.2 M acetic acid until free of tricresol.4.6 g of material of the type obtained by chromatography on CMC asdescribed in the previous section was dissolved in 46 ml 0.2 M aceticacid, filtered through a Millipore filter and allowed to sink into thecolumn. Elution was carried out with 0.2 M acetic acid at a flow rate of50 ml/min. Fractions of ml each were collected, starting with theintroduction of the polypeptide solution into the column. FIG. 2 showsthe optical density of the fractions at 280 u. Fractions 43-48 collectedat the greatest optical density as shown in FIG. 2 and were combined andlyophilized. The lyophilized material weight=1.2 g.

The second CMC column.

50 g CMC was washed as described for the first column but then pouredinto a tube 5X40 cm, and stored under 0.1 M NH Second Chromatography onCMC.

The CMC column was equilibrated with 0.1 M NH HCO After equilibration.it had the dimensions 5X17 cm. 2 g of the material obtained as describedin the preceeding section was dissolved in 7 ml of water, and 7 ml of0.2 M NH followed by ml of 0.1 M NH HCO were added and the pH wasadjusted to 8:01 with 0.1 M NH (about 1 ml). The solution was clarifiedby filtration through a Millipore" filter and allowed to sink into theCMC column. Elution was carried out with 0.1 M NH, HCO at a flow rate of14 ml/min. Fractions of 14 ml each were collected. FIG. 3 shows theoptical density of the fractions at 280' run and 1 cm light path.Fractions 61-83 were collected beginning at approximately 1/4-1/5 thepeak value of the greatest optical density as illustrated in FIG. 3.These fractions were combined and lyophilized. The lyophilized materialweighed 230 mg.

Countercurrent Distribution.

217 mg of the material obtained as described in the preceeding sectionwas subjected to countercurrent distribution in the apparatus (from H.0. Post, lnc.,

Middle village, New York, USA.) with an inert argon atmosphere. Thephase system is butanoI-1/0.l M NH; HCO After completion of the 200 tubetransfer, the two phases in each tube were made to coalesce by theaddition of 3 ml of ethanol. The optical densities of the homogeneoustube contents were determined at 215 mn and 1 cm light path. The valuesobtained are given in FIG. 4. Collection of the contents of tubes 60-85at the first full peakof the optical density curve were combined anddissolved in 20 volumes of water. The pH of the solution was brought to2.5-2.7 with HCl and the polypeptides were recovered from solution by adsorption to alginic acid, elution with 0.2 M H Cl and exchange of.chloride for acetate, followed by lyophilization. Thelyophilizedmaterial'weighed 45 mg. and may be considered the final product althoughfurther purification may be made.

The material obtained by countercurrent distribution above showed onestrong and one faint band when analyzed by polyacrylamidegel-electrophoresis. At least sevenbands were visible in the preparationimmediately preceding the countercurrent step. Analysis for N-terminalamino acid, by the phenylisothiocyanate method revealed only one suchacid, histidine. Tryptophan and cystine/cysteine were absent as shown bythe Voisnet-Rhode dimethylaminobenzaldehyde reaction, and by analysis ofperformic acid-oxidized material. These observations indicated thematerial to be sufficiently pure for work on the structure of VIP. Acidhydrolysis and two-dimensional paper chromatographic analysis of thehydrolysate showed, however, that two of the amino acids in thehydrolysate. glycine and proline, occured in much lower amount than anyof the others. and consequently, were derived from contaminatingpolypeptide material. On chromatography of the highly purifiedpreparation from the countercurrent distribution on Sephadex G-25, fine.in 0.2 M acetic acid (20 mg polypeptide on a 0.9Xl40 em column), thematerial from the fringe fractions of the polypeptidecontaining part ofthe eluate contained distinctly more glycine and proline than materialfrom the middle fractions. Table I shows the results of the analysis ofan acid hydrolysate of 470 micrograms of the latter type THE STRUCTUREOF VIP The following sequence has been established: His (Ala, Asp, Ser,Val). Phe. Thr. (Asx Tyr. Thr. Arg. Leu. Arg (Lys, Glx). Met. Ala (Val,Lys, Lys Tyr. Leu. Asn. Ser. Ile. Leu. Asn. NH

This information is based on the following evidence:

Treatment with cyanogen bromide cleaves the polypeptide into twofragments, one with N-terminal histidine, like the polypeptide itself,obviously the N- terminal part, denoted VIP-CNBr-N, and the other withC-terminal alanine, the C-terminal VIP-CNBr-C. Chymotrypsin cleavesVIP-CNBr-C into three fragments with the composition:

VlP-CNBr-C-N Ala (lys Val). Tyr

VIP-CNBr-C-l Leu (Asn, Ilc, Ser). Leu

and

VlP-CNBr-C-C- Asn. NH;

where -N stands for N-terminal, -I for intermediate and -C forC-terminal.

VIPCNBr-N is cleaved by chymotrypsin into 4 peptides which can bearranged in the order in which they occur in the intact VIP-CNBr-N, withthe aid of the additional information obtained by cleavage of the latterwith trypsin. These four peptides are:

VlP-CNBr-N-N His (Ala, Asp, Ser, Val) Phe VIP-CNBr-N-IN Thr (Asx TyrVlP-CNBr-N-I-C Thr. Arg. Leu

Arg (Lys, Glx). Met

(recovered as homocysteine) BIOLOGICAL ACTIONS OF VIP The newly isolatedpolypeptide VIP has a wide variety of biological actions in severalanimal species. Most of the experiments have been conducted inanesthetized dogs (anesthetic: pentobarbital or chloralose/urethane);some effect have also been confirmed in other species. 1

A. The Cardiovascular System 1. Peripheral (Systemic) Vasodilation Thisis one of the major effects of the peptide and is the basis for bioassaythrought the process of purification. The vasodilatory effect wasmeasured by recording. the femoral arterial blood flow (electromagneticprobe) during the infusion of the peptide into a branch of the samefemoral artery. Flow increased by 50 percent with the infusion of 40ng/kg of the peptide. Vasodilation was also seen in the superiormesenteric and,

hepatic arterial beds following local infusion of the peptide. Infusioninto the renal artery, however, resulted in either no change or anaeutal decrease in blood flow.

2. Blood Pressure Intravenous or intra-arterial infusion of VIP usuallyresulted in a fall in mean systemic arterial blood pressure, asmonitored by a transducer attached to an aortic catheter. Thishypotension was observed with all but the smallest doses; for example,400 ng/kg reduced mean blood pressure by approximately 15 mm Hg. Thehypotensive effect has also been shown in normal rabbits and rats and inthe special breed of spontaneously hypertensive rats.

3. Pulmonary Vasodilation It has been found that the vasodilatory actionof VIP extends also to the pulmonary vessels. This has been demonstratedin isolated lung lobes perfused at a constant flow with Krebs solution.The vasodilatory action is demonstrable even in the presence of normalpulmonary arterial pressure, but is more noticeable if this pressure hasbeen raised previously, as by hypoxic (8 percent oxygen) breathing, orby the infusion of histamine or nor-epinephrine into the same lobe. Thisaction was not mediated by B- adrenergic stimulation or by a-adrenergicblockade.

4. Splanchnic Vasodilation Infusion of VIP into the hepatic or thesuperior mese nteric artery leads to increased blood flow in theseart'eries, despite the associated fall in mean arterial blood pressure.I

5. Cardiac Effects: Positive Inotropic Action A positive inotropicaction for VIP has been demonstrated in two types of preparations;intact, anesthetized dog and isolated papillary muscle from cat or dogheart. In the intact'dog', where heart action was paced electrically,intravenous injections of VIP in doses too small to lower blood pressure(0.05 -0.2' pig/ g)y increased left ventricular dP/dt, even though leftventricular end-diastolic pressure decreased or remained unchanged.

Myocardial contractility, as measured by the force of isometriccontraction of cat or dog papillary muscle, also increased with theapplication of VIP (0.1 #g/rnl This positive inotropic effect of VIP wascomparable to, and often stronger than that of glucagon, but weaker thanthat of isoproterenol.

No chronotropic action was demonstrable on isolated rat atrial muscle.

B. The Respiratory System Besides the (l) vasodilatory action onpulmonary vessels, described above, VIP has the following effects on therespiratory system:

2. Respiratory Stimulation Doses of 0.9 ,ug/kg, infused into commoncarotid artery enhanced respiratory frequency (68 percent), and to alesser extent, tidal volume (0.4 percent), resulting inhyperventilation, a fall in arterial blood P and a rise in arterial P 3.Relaxation of Bronchial Smooth Muscle This action was demonstrated onisolated trachea of guinea-pig, perfused with Krebs solution. Thisrelaxant effect was not affected by ,B-adrenergic blockade.

C. Carbohydrate Metabolism l. Hyperglycemia Infusions of VIP (I Lg/kg,intravenously or intraarterially) indogs led to elevation of plasmaglucose concentrations from I 10 to 140 mg per I ml. The hyperglycemiainduced by the same dose of glucagon was 144 percent greater.

2. Glycogenolysis.

Incubation of VIP with slices of rabbit liver was associated withincreased glycogenolysis, to an extent that was not significantlydifferent from that induced by the same dose of glucagon in the samesystem.

D. Smooth Muscle-Relaxant Activity Addition ofVIP to Krebs solutionsuperfusing isolated smooth muscle preparations caused relaxation ofthese. tissues: rat stomach strip, guinea-pig trachea (mentionedearlier, under H), guinea-pig gallbladder, chick rectum, and chickrectal cecum. In no case was the relaxation affected by antagonists ofhistamine, serotonin acetylcholine, aor B-adrenergic receptors (4).

E. When VIP was infused into a carotid artery of anesthetized dogs, theelectroencephalographic pattern was changed in a manner showing lightersleep, i.e., a tendency to wakefulness. The minimal effective doseswere, however, larger than those required to stimulate respiration ordecrease arterial blood pressure.

THERAPEUTIC APPLICATIONS OF VIP 1. The potent vasodilator actionsuggests possible usefulness in promoting peripheral blood flow inextremities, and in relieving pulmonary hypertension in disease statesassociated with constriction of pulmo-' nary vessels.

2. Because of its hypotensive action, the polypeptide may prove usefulas an additional tool in the management of systemic hypertension.

3. The myocardial-stimulant action may make the polypeptide a usefulagent in the supportive treatment of certain cases of congestive heartfailure. Glucagon has been used for such purposes, and VIP appears to bemore potent in this regard, at least in some cases.

4. The smooth-muscle relaxant properties of the polypeptide render itpotentially useful in the management of excessive contractions ofcertain organs such as gallbladder.

5. The stimulant effect of VIP on respiration and on the central nervoussystem may make it useful as a respiratory stimulant and as an analepticagent.

DOSAGE It is recommended that the dosage to humans or animals be byintravenous injection of 0.02-10 ug VIP/kg of body weight. The carriermay be any physiologically safe and unreactive solvent. Among thoseconsidered useful, are the normal saline solutions, THAM solution, andothers. The concentration of VIP in the carrier is not critical and mayvary widely.

We claim:

1. A method ofextracting and isolating VIP, a new biologically activepolypeptide from the'intestines of mammals comprising:

extracting crude VIP and secretin from said intestines, subjecting saidcrude VIP and secretin to a first ion-exchange chromatography,collecting fractions containing crude VIP after all the secretin hasbeen removed,

subjecting said crude VIP further to gel chromatography, collectingfractions having approximately the greatest optical density measured at280 mp. containing further purified VIP, subjecting the so purified VIPto a second ion-exchange chromatography and collecting fractions afterthe optical density measured at 280 mp. of successive fractions fallsbelow approximately one-fourth of the peak value, and thereafter,subjecting the further purified VIP to countercurrent distributionseparation and collecting purified VIP at approximately those fractionshaving the first full peak value of optical density measured at 215 mu.

2. The method of claim 1 including dissolving said crudeVIP in a bufferat approximately pH 7-9 for said first and second ion-exchangechromatography,

dissolving said crude VIP in an organic acid in said gel chromatography.

3. The method of claim 1 including dissolving said crude VIP in a bufferselected from sodium and ammonium carbonates, bicarbonates, andphosphates and acid phosphates at a pH of 7-9 approximately, prior tothe first ion exchange chromatography, dissolving said crude VIP forsaid gel chromatography in an organic acid, said gel filter beingselected from a cross-linked dextran and cellulose, using analcohol-buffer, as defined above, system to isolate VIP.

4.'The method of claim 3 including said buffers being 'Na HPO for saidfirst ion exchange, NH HCO for said second ion exchange, said organicacid with said gel chromatography being acetic acid and saidcountercurrent distribution separa'tionsystem being l-butanol and NH HCOand said first and second ion-exchange chromatography filter materialbeing CMC.

pH at between 7.8 and 8.2 in said first and second ionexchangechromatography.

1. A METHOD OF EXTRACTING AND ISOLATING VIP, A NEW BIOLOGICALLY ACTIVEPOLYPEPTIDE FROM THE INTESTINES OF MAMMALS COMPRISING: EXTRACTING CRUDEVIP AND SECRETIN FROM SAID INTESTINES, SUBJECTING SAID CRUDE VIP ANDSECRETIN TO A FIRST IONEXCHANGE CHROMATOGRAPHY, COLLECTING FRACTIONSCONTAINING CURDE VIP AFTER ALL THE SECRETIN HAS BEEN REMOVED, SUBJECTINGSAID CRUDE VIP FURTHER TO GEL CHROMATOGRAPHY, COLLECTING FRACTIONSHAVING APPROXIMATELY THE GREATEST OPTICAL DENSITY MEASURED AT 280 M$CONTAINING FURTHER PURIFIED VIP, SUBJECTING THE SO PURIFIED VIP TO ASECOND ION-EXCHANGE CHROMATOGRAPHY AND COLLECTING FRACTIONS AFTER THEOPTICAL DENSITY MEASURED AT 280 M$ OF SUCCESSIVE FRACTIONS FALLS BELOWAPPROXIMATELY ONE-FOURTH OF THE PEAK VALUE, AND THEREAFTER, SUBJECTINGTHE FURTHER PURIFIED VIP TO COUNTERCURRENT DISTRIBUTION SEPARATION ANDCOLLECTING PURIFIED VIP AT APPROXIMATELY THOSE FRACTIONS HAVING THEFIRST FULL PEAK VALUE OF OPTICAL DENSITY MEASURED AT 215 M$.
 1. A methodof extracting and isolating VIP, a new biologically active polypeptidefrom the intestines of mammals comprising: extracting crude VIP andsecretin from said intestines, subjecting said crude VIP and secretin toa first ion-exchange chromatography, collecting fractions containingcrude VIP after all the secretin has been removed, subjecting said crudeVIP further to gel chromatography, collecting fractions havingapproximately the greatest optical density measured at 280 m Mucontaining further purified VIP, subjecting the so purified VIP to asecond ion-exchange chromatography and collecting fractions after theoptical density measured at 280 m Mu of successive fractions falls belowapproximately one-fourth of the peak value, and thereafter, subjectingthe further purified VIP to countercurrent distribution separation andcollecting purified VIP at approximately those fractions having thefirst full peak value of optical density measured at 215 m Mu .
 2. Themethod of claim 1 including dissolving said crude VIP in a buffer atapproximately pH 7-9 for said first and second ion-exchangechromatography, dissolving said crude VIP in an organic acid in said gelchromatography.
 3. The method of claim 1 including dissolving said crudeVIP in a buffer selected from sodium and ammonium carbonates,bicarbonates, and phosphates and acid phosphates at a pH of 7-9approximately, prior to the first ion exchange chromatography,dissolving said crude VIP for said gel chromatography in an organicacid, said gel filter being selected from a cross-linked dextran andcellulose, using an alcohol-buffer, as defined above, system to isolateVIP.
 4. The method of claim 3 including said buffers being Na2HPO4 forsaid first ion exchange, NH4HCO3 for said second ion exchange, saidorganic acid with said gel chromatography being acetic acid and saidcountercurrent distribution separation system being 1-butanol andNH4HCO3, and said first and second ion-exchange chromatography filtermaterial being CMC.
 5. The method of claim 1 including eluting the crudeVIP with acid to obtain the fractions with VIP in the first ion-exchangechromatography, converting said VIP to an acetate and freeze-drying saidcrude VIP product.